SUMMARY Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid storage disorders is not clear. Here we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery, whilst the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-β-cyclodextrin, which is used for cholesterol depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease.

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show the μ-homology domain (μHD) of FCHO1/2 represents a novel endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μHD interacts with the Bmp receptor Alk8, uncovering a new endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus contradict the primacy of FCHO1/2 in coat initiation.

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.

It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.Gap junctions are conglomerations of intercellular channels that permit the direct exchange of ions, second messengers, and other molecules of less than 1500 Da between the cytoplasmic interiors of contiguous cells. The channels are composed of a family of proteins called connexins (Cx) 2 that are designated according to their molecular mass (1). The assembly of Cxs into GJs is a multistep process. The newly synthesized Cxs are cotranslationally inserted into the endoplasmic reticulum, which oligomerize to form hexamers called connexons. The connexons are delivered to the cell surface via Golgi and trans-Golgi network in small vesicles and dock with the connexons in the contiguous cell membranes to form intercellular channels. A GJ plaque is formed when several intercellular channels cluster (2, 3). Because of the short half-life of Cxs (2-5 h), the plaque is in a dynamic state and is thought to model and remodel constantly through recruitment of newly synthesized connexons to the periphery and through endocytosis of moribund plaque components from the center or through the endocytosis of the plaque in its entirety (4 -7). Although much has been learned about the intracellular transport of Cxs, the delivery of connexons to the cell surface, the formation of cell-to-cell channels, the spatio-temporal events and cellular factors required for the initial docking of connexons, the de novo formation of a nascent GJ plaque, its growth, and disassembly are poorly understood (2,8).The proximity between the plasma membranes of adjoining cells is, a priori, necessary for the formation of GJs, and cell-cell adhesi...